ES2807897T3 - Stem cell therapy based on adipose stem cells - Google Patents

Abstract

A composition comprising a suspension of a substantially homogeneous and immunosuppressive population of adult human adipose tissue (ASC) stem cells in a protein-free cryoprotectant at a concentration of at least 1.5 x 107 cells per ml.

Description

DESCRIPTION

Stem cell therapy based on adipose stem cells

Field of the invention

The present invention relates to adipose tissue stem cells (ASCs) and compositions, as well as to methods for preparing and using such ASCs and compositions for therapy.

Background of the invention

A multitude of previous clinical studies have established that mesenchymal stromal cells (MSCs) from bone marrow as well as from adipose tissue have significant regenerative abilities. Mesenchymal stromal cells from both tissues of origin enhance regeneration through paracrine mechanisms, releasing extracellular substances that promote natural endogenous repair mechanisms including matrix remodeling, revascularization, and immune modulation.

MSCs have been shown to be safe and effective in the treatment of severe stable coronary artery disease and refractory angina and chronic ischemic heart disease (eg, Mathiasen et al., 2012; Mathiasen et al., 2013). The clinical safety of treating chronic myocardial ischemia with ASC has also been documented (Qayyum et al., 2012; Ekblond, 2015). MSCs also have immunosuppressive properties, stemming from their ability to inhibit or arrest dendritic cell maturation and proliferation of T lymphocytes, B lymphocytes, and NK cells, and are being explored for the treatment of various autoimmune or other inflammatory disorders (Gebler et al., 2012; Wang et al., 2014).

However, current ASC production methods and clinical logistics are less than optimal, preventing a wide diffusion of this type of treatment. Thus, there is a need for safe and effective methods to produce and preserve high quality allogeneic ASC preparations suitable for a wide range of therapeutic applications.

Document WO 2014/203267 (Kaziak Research PVT Ltd.) refers to a method for the isolation, purification and expansion on an industrial scale of MSCs from human adipose tissue and their use in the treatment of type 1 diabetes mellitus, critical ischemia of limbs and other disorders.

Document WO 2006/037649 (Cellerix S.L. and the Autonomous University of Madrid) refers to the identification and isolation of multipotent cells from non-osteochondral mesenchymal tissue, characterized by certain markers.

Despite these and other advances in the art, there is still a need for new manufacturing and formulation technologies for ASC.

Summary of the invention

The present inventors have discovered that high quality "ready-to-use" ASC preparations can be efficiently produced and frozen at a high concentration in a protein-free cryoprotectant. Frozen ASC preparations are, when thawed, ready for clinical use. Furthermore, ASC preparations have immunosuppressive properties, making them suitable for both autologous and allogeneic use, for example, in immunosuppressive therapy.

Therefore, in a first aspect, the present invention relates to a process that prepares a pharmaceutical composition comprising a substantially homogeneous population of adult human stem cells, comprising the following steps:

(i) adding the stromal vascular fraction (SVF) of a lipoaspirate collected from a donor to a bioreactor where at least one surface is pretreated to promote adhesion of adult human stem cells;

(ii) in the bioreactor, culturing adherent cells for confluence in serum-free culture medium supplemented with human platelet lysate;

(iii) separate adherent cells;

(iv) freezing the separated cells in a protein-free cryoprotectant at a concentration of at least 1 x 10 6 cells / ml;

(v) thaw the frozen cells and repeat steps (ii) and (iii), and optionally (iv), at least once, (vi) freeze the separated cells at a concentration of at least 1.5 x 107 cells / ml; Y

(vii) optionally thawing the frozen composition.

In a second aspect, the invention relates to a composition, such as a pharmaceutical composition, which It comprises a suspension of a substantially homogeneous and immunosuppressive population of adult human stem cells derived from adipose tissue in a protein-free cryoprotectant at a concentration of at least 1.5 x 107 cells per ml. The composition is optionally frozen. In one embodiment, the composition is prepared using the process of the first aspect.

In one embodiment, at least about 80% of the ASC population expresses CD90, CD73, CD13, CD105, CD29, CD166, CD10, CD140b, CD160, CD204, CD272, CD44, CD49a, CD54, CD9, Galectin 3, Galectin 9, HLA-G and LTp3R and at most approximately 15% of the ASC population expresses CD45, CD19, CD14, CD106, CD31 and CD36.

In a third aspect, the invention relates to the use of said composition as a medicine, for example, for immunosuppression, for the treatment of an autoimmune disorder or other inflammatory disorder, and for the treatment of ischemic disorders or other disorders characterized by the destruction of tissue. In one embodiment, the composition is used in a method of treating ischemic heart disease, typically administering the composition by direct intramyocardial injection. Allogeneic therapy is particularly contemplated, that is, where the donor of the ASC is not the patient to whom the composition will be administered.

These and other aspects and embodiments are explained in more detail below.

Legends of the figures

Figure 1 represents ASC production processes according to some embodiments of the invention.

Detailed disclosure of the invention

The present invention relates to a stem cell product based on ASCs isolated from healthy donors, typically by two rounds of ASC expansion in a bioreactor separated by a cryopreservation step, resulting in a composition suitable for cryopreservation in a bank. of cells. The product is useful as an allogeneic therapeutic drug, for example, for regenerative therapy in disorders or diseases characterized by ischemia or other tissue destruction, such as heart disease with and without heart failure, for immunosuppression of autoimmune reactions or transplant rejection, or therapy. anti-inflammatory of inflammatory diseases. In particular, the ASC composition can be used as a ready-to-use cryopreserved product, stored in, for example, liquid nitrogen, and ready to use directly after thawing. The ASC composition can be administered intravenously, intraarterially, or by direct infusion or injection into tissue, eg, myocardium.

On the other hand, a cell bank comprising multiple ASC preparations from different donors according to the invention can provide personalized treatment, for example by allowing tissue matching between donor and recipient prior to treatment, various recipient treatments and, in case the recipient needs multiple treatments, the ability to switch ASC from one donor to another. The latter is particularly useful in case the recipient develops an alloantibody response to ASCs from a previously administered ASC preparation.

Definitions

"ASC", "stem cells derived from adipose tissue", "stromal cells derived from adipose tissue" and the like refer to multipotent stromal stem cells, also known as mesenchymal stem cells, multipotent stromal cells, multipotent stem cells, and stromal cells. / stem mesenchymal, which come from adipose tissue. Certain criteria for identifying ASC are known in the art and are described in, for example, Bourin et al. (2013). In some embodiments, ASCs are characterized by their ability to differentiate along adipocyte, chondroblastic, and osteoblastic lineages under appropriate conditions. ASCs in culture can be characterized by the expression of one or more of the following cell surface markers: CD90, CD73, CD105 and the lack of expression of CD45 and CD31. In some embodiments, they can be distinguished from bone marrow-derived MSCs by their positivity for CD36 and negativity for CD106.

The stem cell population prepared according to the inventive method described herein is "substantially homogeneous", meaning that most of the cells meet the ASC standards. Typically, a substantially homogeneous ASC population in a composition according to the present invention is characterized by at least about 80% of the ASC population expressing CD90, CD105, CD13, CD73, CD166, CD29, and optionally CD10. , CD140b, CD160, CD204, CD272, CD44, CD49a, CD54, CD9, Galectin 3, Galectin 9, HLA-G and LTpR; and by at most approximately 15% of the ASC population that expresses CD45, CD31, CD14 and CD19. In some ASC populations of the invention, one or more of CD90, CD73, CD13, CD105, CD29, CD166, CD10, CD140b, CD160, CD204, CD272, CD44, CD49a, CD54, CD9, Galectin 3, Galectin 9, HLA -G and LTpR can be expressed as at least about 80%, such as at least about 85%, such as at least about 90%, such as at least about 95%, such as at least about 97% or more of the ASC population. Similarly, in some ASC populations of the compositions of the invention, one or more of CD45, CD19, CD14, CD106, CD31 and CD36 can be expressed at most about 15%, such as at most about 12%, such as at most about 10%, such as at most about 7%, such as at most about 5%, such as at most about 3% or less of the ASC population. The specific ranges contemplated for these and other markers are those defined by the minimum and maximum expression percentages of an ASC population as shown in Tables 15 and 20.

By "adipose" is meant any fatty tissue. The adipose tissue can be brown or white adipose tissue, originating from the abdominal area or another site of adipose tissue. In certain embodiments, the adipose is subcutaneous white adipose tissue or visceral adipose tissue or any other tissue that contains adipose cells. The adipose tissue can be from any mammal. Preferably, the adipose tissue is human, most preferably from an adult human. A convenient source of adipose tissue is liposuction surgery.

A "liposuction", as used herein, refers to material removed during liposuction through an aspirator, ie, a suction device. The lipoaspirate comprises adipocytes, fat, connective tissue, blood vessels, and a stromal vascular fraction. Any type of liposuction method known in the art can be used, including, but not limited to, suction-assisted, ultrasound-assisted, power-assisted, double-cannula-assisted, laser-assisted, and water-assisted liposuction (WAL, for its acronym in English). The WAL is, however, one of the preferred options. The "stromal vascular fraction" or "SVF" can be isolated after liposuction using methods known in the art, and exemplified below.

As used herein, the term "bioreactor" refers to any device in which biological and / or biochemical processes are performed under monitored and controlled environmental and operating conditions, eg, pH, temperature, gas supply / air and nutrients and waste disposal.

The term "cryopreserve" or its various grammatical forms, as used herein, refers to the preservation of cells for storage in a cryoprotectant at subzero temperatures. For long-term storage, cryovials containing the cells and cryoprotectant are generally placed in liquid nitrogen.

The term "cryoprotectant", as used herein, refers to an agent that minimizes the formation of ice crystals in a cell or tissue, when the cell or tissue is cooled to sub-zero temperatures and results in damage. substantially less cell or tissue after heating compared to cooling effect without cryoprotectant.

"Viability", as used herein, refers to the characteristic of cells not to absorb membrane waterproofing dye (eg, Trypan blue, FVS-780, SYTOX blue, propidium iodide), which demonstrates the integrity of the cell membrane.

"Proliferation capacity", as used herein, refers to the ability of cells to multiply in a suitable culture medium. Proliferation capacity can, for example, be represented by the relative number of cells after a culture period of 24 hr, 48 hr or 72 hr compared to the number of cells initially plated. This can also be expressed as "population doublings" over a certain period. For example, a population doubling of at least 1 for 48 h in cell culture means that the number of seeded cells has doubled at least once during that period.

As used herein, the term "donor" refers to the human or mammal from whom adipose tissue is removed, typically by liposuction. Preferably, the human is an adult.

The terms "treatment", "therapy" and the like are used herein to refer generally to obtaining a desired pharmacological and / or physiological effect. The effect can be prophylactic in terms of the complete or partial prevention of a disease or a symptom thereof and / or it can be therapeutic in terms of stabilization or partial or complete cure for a disease and / or an adverse effect attributable to the illness. "Treatment", as used herein, covers any treatment of a disease of a mammal, particularly a human or veterinary subject, and includes: (a) preventing the onset of the disease or symptom in a subject who may be predisposed the disease or symptom, but has not yet been diagnosed with it; (b) inhibiting the disease symptom, that is, arresting its development; or (c) alleviating the disease symptom, ie, causing regression of the disease or symptom.

In the context of the therapeutic use of the disclosed pharmaceutical compositions, in 'allogeneic' therapy, donor and recipient are genetically different individuals of the same species, while in 'autologous' therapy, donor and recipient are the same individual.

The terms "recipient", "subject" and "patient" are used interchangeably herein and refer to the mammalian subject for whom treatment or therapy is desired, in particular, humans.

Specific embodiments of the invention

Process:

The process according to the invention offers a safe and efficient manufacturing technology based on, for example, the combination of human platelet lysate as a growth supplement for ASC, expansion in a closed bioreactor system and final formulation of ASC as a product. Ready-to-use cryopreserved allogeneic with high quality ASC.

An overview of the process according to some different embodiments is shown in Figure 1.

Typically, using the process of the invention, the preparation of a batch of ASC product from an SVF only takes about 15 to about 18 days, excluding time in freezing. The expansion efficiency is particularly surprising, considering that an average yield of 11 ± 5 vials of intermediate products can be obtained from an SVF test in the bioreactor (average of 3 SVF tests), each of which generates an average batch yield of 5 ± 2 ampoules of final product (based on an average of 8 ASC bioreactor tests). Thus, an average yield of 55 cryovials with approximately 110 million ASCs in each can be obtained from approximately 100 million mononuclear cells (MNCs) in the SVF. Additionally, as described in the Examples, the ASC product is characterized by high viability (average 90 ± 2%), as determined immediately after thawing the ASC product from the second step.

In one embodiment, the process for preparing a pharmaceutical composition comprising a substantially homogeneous population of adult human stem cells, comprises the steps of

(i) adding the SVF from a lipoaspirate collected from a donor to a bioreactor where at least one surface is pretreated to promote adhesion of adult human stem cells;

(ii) in the bioreactor, culturing adherent cells for confluence in serum-free culture medium supplemented with human platelet lysate;

(iii) separate adherent cells;

(iv) freezing the separated cells in a protein-free cryoprotectant at a concentration of at least 1 x 10 6 cells / ml;

(v) thaw the frozen cells and repeat steps (ii) to (iii) at least once,

(vi) freeze the separated cells at a concentration of at least 1.5 x 107 cells / ml; Y

(vii) optionally thawing the frozen composition.

The disclosure also enables a process for preparing a composition comprising a substantially homogeneous population of adult human stem cells, comprising the steps of

(i) adding the SVF from a lipoaspirate collected from a donor to a bioreactor where at least one surface is pretreated to promote adhesion of adult human stem cells;

(ii) in the bioreactor, culturing adherent cells for confluence in serum-free culture medium supplemented with human platelet lysate;

(iii) separate adherent cells;

(iv) repeat steps (ii) and (iii) at least once;

(vi) freezing the separated cells at a concentration of at least 1x107 cells / ml; and optionally (vi) thawing the frozen composition.

In one embodiment, the process for preparing a pharmaceutical composition comprising a substantially homogeneous population of adult human stem cells comprises the steps of

(i) adding the stromal vascular fraction (SVF) of a lipoaspirate collected from a donor to a bioreactor where at least one surface is pretreated to promote adhesion of adult human stem cells; (ii) culturing adherent cells of the SVF for confluence in serum-free culture medium supplemented with human platelet lysate;

(iii) separate adherent cells;

(iv) freezing the separated cells in a cryoprotectant at a concentration of at least 1x106 million cells / ml;

(v) thawing the frozen cells and repeating steps (ii) to (iv), freezing the separated cells at a concentration of at least 1.5 x 107 cells / ml; and optionally

(vi) thawing the frozen composition.

SVF is isolated from a lipoaspirate obtained from a healthy donor, eg, 50 ml, 100 ml, 200 ml, 300 ml, or 500 ml, such as between 100-300 ml, of lipoaspirate. Typically, adipose tissue is first separated from non-adipose tissue using a tissue collection container that uses decantation, sedimentation, or centrifugation techniques to separate the materials. The adipose tissue can then be disaggregated using methods such as mechanical force (pitting or shear forces), enzymatic digestion with one or more proteolytic enzymes, such as collagenase, trypsin, TrypLe Select, lipase, HI liberase, pepsin, or a combination of mechanical and enzymatic methods. After that, the remaining cells can be recovered by filtration, centrifugation or the like. Examples of methods for recovering SVF from a lipoaspirate are described in Godthardt, et al. (2008) MACS Miltenyi Biotec Information Pamphlet and WO 2014/138383.

In one embodiment, approximately 100 ml of liposuction is obtained from a donor by liposuction of the abdomen under local anesthesia. The lipoaspirate is washed twice with phosphate buffered saline (PBS) pH 7.4 to remove residual blood. The adipose tissue is then digested by incubation with collagenase dissolved in a balanced salt solution at 37 ° C for 45 minutes in constant rotation. Collagenase is neutralized with medium containing 5% human platelet lysate and 1% penicillin / streptomycin and filtered through a 100 pm filter. The remaining cells are centrifuged at 1200 x g for 10 minutes at room temperature, resuspended and counted using a cell counter according to the manufacturer's instructions.

At least one surface of the bioreactor is pretreated to facilitate or promote ASC adhesion, either by the bioreactor manufacturer or at some chosen time point before starting the production process. Various types of treatments are known in the art to promote cell adhesion and include, for example, tissue culture treatment and coating with charged synthetic polymers, nanofibers, mucopolysaccharides, and various protein compositions. For tissue culture treatment, a polystyrene-based surface in the bioreactor is modified with plasma gas, which results in the hydrophobic plastic surface becoming more hydrophilic, promoting net negative charge cell attachment. As for the precoating of the surface, the protein compositions useful for this purpose may comprise one or more plasma proteins such as, for example, fibrinogen, fibronectin, Factor VIII, von Willebrand factor and Factor XIII; one or more extracellular matrix proteins such as, for example, collagens and laminins; and / or one or more proteoglycans. In one embodiment, the protein composition comprises or consists of one or both of fibrinogen and fibronectin. In one embodiment, the protein composition comprises or consists of cryoprecipitate. Cryoprecipitate is a well known blood product prepared from plasma, for example, where fresh plasma is frozen and thawed and the precipitate is collected. The product typically contains fibrinogen and Factor VIII, as well as, for example, von Willebrand factor, Factor XIII, and fibronectin. In some embodiments, the cryoprecipitate contains at least 140 mg or more of fibrinogen per 70 IU of Factor VIII, optionally prepared from AB or low A blood donors. In another embodiment, the protein composition comprises or consists of lysate of human platelets, described below.

Basic classes of bioreactors suitable for use in the process of the present invention include hollow fiber bioreactors and roll, spin, or roll perfusion systems, with or without microcarriers or discs, suitable for anchor-dependent cell expansion. Preferably, the bioreactor is a functionally closed system or protected by sterile barrier filters, capable of providing a continuous supply of culture medium and continuous debris removal during cell culture. Most preferred are disposable hollow fiber bioreactors enclosed in an incubator, which provide a surface area for cell attachment of at least 0.5 m2, such as at least 1 m2, such as at least 1.5 m2, such as at least 2 m2, such as between 1 to 3 m2. Preferably, the surface area is at least 2 m2, such as about 2.1 m2. An example of such a bioreactor is the quantum cell expansion system (in this document also called "quantum bioreactor") which is fed through two circulation circuits with inlets for media and reagents or cells, the waste being disposed of in a bag of waste. As shown in Example 7, expansion of ASCs in a bioreactor significantly increased expansion rate and throughput relative to manual processing in standard tissue culture flasks.

Prior to loading the SVF (or first pass ASCs) into the bioreactors, the system can be primed with a buffer, eg phosphate buffered saline, and subsequently loaded with a protein or other coating composition. Before loading cells, the buffer can be washed from the system and replaced with complete medium.

The cells can then be added to the bioreactor. For example, approximately 10, 20, 50, 100, 200, or 500 million mononuclear cells (MNCs) can be loaded from the SVF preparation or approximately 5, 10, 20, 50, or 100 million ASCs from the first step in the Bioreactor primed and coated through the inlet, optionally through a filter. Preferably, for MNCs, about 100 million cells of SVF are loaded. For the second pass, preferably, about 5 to 50 million cells of the first pass ASCs are loaded, such as between 5 and 30, 5 and 25, 10 and 30 or between 15 and 25 million cells. For any higher step of the cells (i.e. 3rd step, 4th step, etc.), the cells can be added in similar amounts to the second step. The cells are then allowed to adhere for a sufficient period of time, such as for at least 5 hrs and / or up to about 24 hrs, after which continuous media feeding is activated. For example, the medium feed rate can start at about 0.1 ml / min, and then be adjusted based on glucose and / or lactate measurements and / or cell expansion.

Any standard cell culture medium can be used, such as, for example, Modified Eagle's Medium from Dulbecco (DMEM), Alpha Minimal Essential Medium (a-MEM). As shown in Example 6, however, the use of a human plasma lysate (HLP) was clearly a more efficient growth supplement in terms of proliferative capacity than the use of FBS, without compromise genomic stability. HPL is normally a cloudy, light yellow liquid that is obtained from human blood platelets after one, two, three, or more freeze / thaw cycles. These cycles cause platelets to lyse, releasing their intracellular contents, including growth factors and the like, into the surrounding medium. Some HPL preparations include blood clotting factors, in which case it may be advantageous to add an anticoagulant such as heparin to prevent clotting. Other HPL preparations can be processed to remove or otherwise inhibit the effect of clotting factors. HPL preparations, some of which are GMP grade, are commercially available from, for example, Compass Biomedical, Inc., Cook General BIotechnologyl, Macopharma SA, Cook Regentech, Mill Creek, iBiologics and Trinova Biochem GmbH under the lines of PLUS, Stemulate, Human Platelet Lysate, PLTMax, XcytePlus products and CRUX RUFA media supplements. Preferably, the culture medium for ASCs comprises from about 1% to about 20%, such as from about 2% to about 15%, such as from about 3% to about 12%, such as from about 5% to about 10%, such as about 5%, 8%, or 10% HPL. Preferably, the culture medium comprises from about 2% to about 15% HPL in, for example, MEM. A preferred HPL preparation is Stemulate, which does not require the addition of heparin (WO 2015031465 A1).

Once cell growth has reached or nearly reached its stationary phase, as determined by, for example, stagnation in glucose consumption and / or lactate production, the ASCs are harvested, usually by loading TrypLe Select into the system. . The collected cells can then be washed and transferred to centrifuge tubes, granulated, and counted.

The first pass ("intermediate") ASCs can then be cryopreserved or alternatively loaded directly into the precoated bioreactor for a second (or 3rd, 4th, etc.) round of expansion. For cryopreservation, intermediate ASCs are suspended in cryoprotectant at a concentration of at least about 1 x 106 cells per ml, such as at least about 2 x 106 cells per ml, at least about 5 x 106 cells per ml, at least about 10x106 cells per ml, at least about 15x106 cells per ml, at least about 20x106 cells per ml, or at least about 50x106 cells per ml, such as between 1x106 and 50x106 cells per ml, such as between 1x106 cells or 20x106 cells per ml.

Second (or higher, such as 3rd, 4th, etc.) ASCs can be cryopreserved. For cryopreservation, the second step (or higher) ASCs are suspended in cryoprotectant at a concentration of 1.5 x 107 cells per ml, at least about 2 x 107 cells per ml, at least about 2.5 x 107 cells per ml, at least about 3x107 cells per ml, at least about 5x107 cells per ml, or at least about 10x107 cells per ml, such as between 1x107 and 5x107 cells per ml, such as between 2 x 107 cells or 3 x 107 cells per ml. In one embodiment, the ASCs from the second step (or higher, such as the 3rd, 4th, etc.) are suspended in cryoprotectant at a concentration of about 2 x 107 cells per ml of cryoprotectant, such as about 2.2 x 107 cells. per ml of cryoprotectant. As used herein, unless the context contradicts it, 2 x 107 cells per ml includes or corresponds to 1.6 x 107 to 2.4 x 107 cells per ml and approximately 2.2 x 107 cells per ml includes or corresponds to 2.0 to 2.4 cells per ml.

The cryoprotectant is preferably protein-free, endotoxin-free, and sterile. While several suitable cryoprotectants are available, non-limiting examples of cryoprotectants contemplated for the ASC compositions of the present invention are CryoStor® (BioLife Solutions), which includes CryoStor CS2, CryoStor CS5 and CryoStor CS10; and ProFreeze (Lonza). CryoStor Freezing Media is sterile, free of serum, protein and animal components, with a pH of 7.5 to 7.7 and an endotoxin level below 1 EU / ml. In one embodiment, the cryoprotectant is Hypothermosol® (CMS, Rockville, Md.) Plus 10% DMSO (WO 2000/002572 A1). Hypothermosol® comprises Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid), Na +, K +, Ca2 +, Mg2 + 1Cl ", H2PO4", HEPES, lactobionate, sucrose, mannitol, glucose, dextran -40 (ie dextran with an average MW of 40,000 Da), adenosine and glutathione (WO 2010/064054 A1). According to the manufacturer, ProFreeze should be supplemented with 10% DMSO at the time of use. For this, reference is made to WO 2000/002572 A1 and WO 2010/064054 A1.

In any embodiment herein where DMSO is used, the DMSO can be replaced by a glucan such as, for example, dextran, having an average molecular weight in the range of 35000 to 45000 Da, such as, for example, dextran. -40.

In one embodiment, the cryoprotectant comprises between 5% and 15% DMSO, such as about 5%, about 6%, about 8%, about 10%, about 12%, or about 15% of DMSO, and Trolox, Na +, K +, Ca2 +, Mg2 + 1Cl-, H2PO4 ", HEPES, lactobionate, sucrose, mannitol, glucose, dextran-40, adenosine, and glutathione. Preferably, the cryoprotectant comprises about 10% DMSO.

In one embodiment, the cryoprotectant comprises a mixture of 1:10 to about 1:20 DMSO and an aqueous solution comprising

(a) one or more electrolytes selected from the group consisting of potassium ions at a concentration ranging from about 35-45 mM, sodium ions ranging from about 80-120 mM, magnesium ions ranging from about 2-10 mM and calcium ions ranging from about 0.01-0.1 mM;

(b) a macromolecular oncotic agent that is large enough to limit escape from the circulation system and effective to maintain an oncotic pressure equivalent to that of blood plasma and selected from the group consisting of human serum albumin, polysaccharide, and colloidal starch;

(c) a biological pH buffer effective under physiological and hypothermic conditions;

(d) a nutritionally effective amount of at least one simple sugar;

(e) an effective amount of mannitol that scavenges waterproof and hydroxyl radicals;

(f) a waterproofing anion impermeable to cell membranes and effective to counteract cell swelling during cold exposure, said waterproofing ion being at least one member selected from the group consisting of lactobionate, gluconate, citrate, and glycerophosphate;

(g) an effective substrate for ATP regeneration, said substrate being at least one member selected from the group consisting of adenosine, fructose, ribose, and adenine; Y

(h) glutathione.

In one embodiment, the cryoprotectant comprises a mixture of 1:10 to about 1:20 DMSO and an aqueous solution comprising

to) . one or more electrolytes selected from the group consisting of potassium ions at a concentration ranging from 35-45 mM, sodium ions ranging from 80-120 mM, magnesium ions ranging from 2-10 mM, and calcium ions ranging they range from 0.01-0.1 mM;

b). a macromolecular oncotic agent that is large enough to limit escape from the circulation system and effective to maintain an oncotic pressure equivalent to that of blood plasma and selected from the group consisting of human serum albumin, polysaccharide, and colloidal starch;

c). a biological pH buffer effective under physiological and hypothermic conditions;

d). a nutritionally effective amount of at least one simple sugar;

e). an effective amount of mannitol that scavenges waterproof and hydroxyl radicals;

f). a waterproofing anion impermeable to cell membranes and effective to counteract cell swelling during cold exposure, said waterproofing ion being at least one member selected from the group consisting of lactobionate, gluconate, citrate, and glycerophosphate;

g). an effective substrate for ATP regeneration, said substrate being at least one member selected from the group consisting of adenosine, fructose, ribose and adenine and

h). at least one agent that regulates apoptotic-induced cell death.

The ASC suspension from the first, second (or higher) step in the cryoprotectant is then added to cryovials. For first-pass (intermediate) ASCs, each cryovial may contain about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 15, or about 20 mL suspension of ASC, corresponding to a number of ASCs in the range of 5 million cells to 250 million cells, such as, for example, about 20, about 30, about 40, about 50, about 60, about 80 or approximately 100 million cells. Preferably, first pass ASC cryovials contain approximately 50 million cells in 5 ml.

For final pass ASCs, i.e. 2nd pass or higher ASCs, each cryovial may contain about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 15 or about 20 ml of ASC suspension, corresponding to a number of ASCs in the range of 10 million cells to 500 million cells, such as, for example, about 40, about 60, about 80, about 100, about 110, about 120, about 160, or about 200 million cells. Preferably, second pass ASC cryovials contain from about 100 million to about 120 million cells in about 5 ml, such as about 110 million cells in about 5 ml. In the compositions of the invention, unless the context contradicts it, approximately 110 million cells include 100 million to 120 million cells.

Freezing can be advantageously carried out by automatic freezing with the use of a controlled speed freezer. After freezing, the vials can be transferred and stored at a temperature in the range of -70 ° C to -196 ° C, such as between -150 ° C and -190 ° C, such as in the range of -180 ° C. Various means are known for freezing and maintaining vials under such freezing conditions, many of which involve liquid nitrogen. They include, for example, immersing the vials in liquid nitrogen, storing the vials in the vapor phase of liquid nitrogen, and placing the vials in so-called dry storage. In the latter type of storage, the vials are not in direct contact with nitrogen in liquid or vapor phase since Nitrogen is contained in the cover of the container, which results in a storage temperature in the range of -180 ° C.

In one aspect, a two-tier cell bank system is established for the vials, which constitutes a working cell bank containing intermediate ASCs from the first pass bioreactor expansions and a cell product bank containing ASC compositions. finally formulated and packaged. In the cell bank, the intermediate ASCs from the first pass are typically cryopreserved at about 50 million cells in about 5 ml of CryoStor10 in cryovials until loading for the second bioreactor expansion. The final ASC product is typically cryopreserved at approximately 110 million cells in approximately 5 ml of CryoStor10 in cryovials until just prior to use. This product may also be designated "CSCC_ASC". In one embodiment, the cell bank comprises a plurality of vials stored under frozen conditions, each vial comprising approximately 5 ml of the ASCs from the second step, where the cell concentration is 2 x 107 cells per ml, for example, in the range of 1.6 x 107 to 2.4 x 107 cells per ml, or about 2.2 x 107 cells per ml, for example, in the range of 2.0 x 107 to 2.4 x 107 cells per ml.

ASC:

ASCs are characterized by their multipotent capacity, marker profile and / or by functional characteristics of ASCs, such as proliferation capacity, viability, recovery and immunosuppressive capacity, even after cryopreservation. These characteristics of the ASCs, detailed below, also apply to the ASCs obtained according to the process of the invention. Marker profiles can, for example, be conveniently determined by flow cytometry using fluorescently labeled antibodies against each marker, eg, as described in Example 2, 10, 11 or 14.

ASCs are characterized, in particular, by their ability to differentiate along adipocyte, chondroblastic, and osteoblastic lineages under appropriate conditions. As shown in Example 4, ASCs prepared according to the manufacturing methods described in Example 1 differentiated into adipocytes, chondrocytes, and osteoblasts when cultured in differentiation medium.

ASCs can also be, or alternatively, characterized according to their phenotype, that is, the marker profile, with respect to their expression of markers in common with other mesenchymal stem / stromal cells, including CD90, CD73, CD105 and CD44, and maintain low or negligible expression levels of CD45 and CD31 (Bourin et al., 2013).

In some embodiments, a substantially homogeneous population of ASC is one where at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 98 % of the stem cell population express CD105, CD90, CD73, CD29 and CD13, and maximum 10%, such as maximum 5%, such as maximum 3%, such as maximum 2% express CD45 , CD34, HLA-DR, CD19 and CD14. Preferably, at least 95% express CD90, CD73 and CD13 and at most 5% express CD45, CD34, HLA-DR, CD19 and CD14.

In some embodiments, a substantially homogeneous population of ASC is one where at least 90% express CD90, CD73, CD13, CD29, and CD166; a maximum of 5% expresses CD45, CD19, CD14 and CD31; a maximum of 10% expresses CD106; between 2 and 15% express CD36; at least 10% express CD146; at least 80% express CD105 and at most 40% express CD34. In some embodiments, at least 95% express CD90, CD73, CD13, Cd 29, and CD166. Furthermore, at least 80%, such as at least 85%, such as at least 90%, such as at least 95%, such as at least 98% can express CD44.

In some embodiments, a substantially homogeneous population of ASC is one where at least about 80%, such as at least about 85% express CD105, CD90, CD73, CD13, CD29, and CD166, such as at least about 90% , such as at least about 95% expressing CD90, CD73, CD13, CD29, and CD166; at most about 15%, such as at most about 10% expresses CD45, CD19, CD14, CD106, CD31 and CD36, such as at most about 7%, such as at most about 5%, such as at most approximately 3% express CD45, CD19, CD14, CD106 and CD31; at least about 2%, such as at least about 5%, such as between about 1% and about 20%, such as between about 2% and about 15%, or between about 5% and about 15% express CD36; at most about 50%, such as at most about 40%, such as at most about 20%, such as at most about 10% expresses CD34; and at least about 10%, such as at least about 12% express CD146. In one embodiment, at least 95% express CD90, CD73, CD13, CD29, and CD166; at least 85% express CD105, at most 2% express CD45, HLA-DR, CD19, CD14 and CD31; between 2% and 15% express CD36; and between 10% and 60% express CD146. In one embodiment, a substantially homogeneous population of ASC is one where the minimum and maximum expression percentage ranges of the markers are as shown in Table 20.

When determined immediately after thawing of the cryopreserved 2nd step cells, at least about 80%, such as at least 85%, such as at least 90%, such as at least 95%, such as at less than 98% of the cells are viable, as determined by dye exclusion (see, for example, Example 3). Preferably, at least 90% of the cells are viable.

Regarding proliferation capacity, when placed in culture immediately after thawing, ASCs are characterized by a population doubling (PD) of at least 1, such as at least 1.3 , such as at least 1.5, such as at least 1.7, such as at least 2, when cultured in tissue culture flasks for 48 h (eg according to the method in Example 3). Preferably, the ASCs have a PD of at least 1, such as at least 1.5. PD is calculated as Ln (N) / ln 2, where N = Cell harvested / Cell seeded.

As shown in the Examples, ASCs are further characterized by their immunosuppressive properties. For example, ASCs may be characterized by one or more or all of the following: suppression of dendritic cell (DC) activation, suppression of peripheral blood mononuclear cell (PBMC) proliferation English), cell surface markers indicative of immunomodulation, especially immunosuppression, or by a change in one or more cell surface markers in response to a cytokine such as interferon gamma.

In one embodiment, ASCs suppress DC activation, for example, by reducing the expression of CD40, CD80, CD86, and HLA-DR by DC mixed with ASC compared to DC not mixed with ASC (ie, a positive control). In a specific embodiment, the assay of Example 9 is used, where ASC and DC are seeded to result in a ratio of about 1: 1; DC are stimulated with 1 pg / ml of lipopolysaccharide (LPS) and 20 ng / ml of interferon gamma and incubated for 24 h; and the respective expression level of CD40, CD80, CD86 and HLA-DR is reduced, on average, to at most 80%, 65%, 70% and 80%, respectively, of the positive control.

In one embodiment, ASCs suppress PBMC proliferation, eg, as determined in a mixed lymphocyte reaction (MLR). This type of assay is well known in the art and may comprise mixing ASC with stimulated PBMC from an allogeneic donor in different ratios, for example in the range of 1:20 to 1: 1, using PBMC without ASC as positive controls, and measuring, after a co-cultivation period of 4 days, the incorporation of 3H-thymidine PBMC (25 pSi / ml) during an incubation period of 18-20 h. Using this type of assay, compared to the positive control, a 1:20, 1:10, 1: 5, and 1: 1 ratio of ASC to PBMC can result in an average 3H-thymidine incorporation of at most about one 80%, 75%, 55% and 25%, respectively, of the positive control.

In some embodiments, ASCs are also or alternatively characterized by specific markers indicative of immunomodulation, especially immunosuppression, such as CD10, CD140a, CD160, CD204, CD258, CD270, CD272, CD44, CD49a, CD54, CD9, Galectin 3, Galectin 9 , HLA-G, LTpR, and combinations thereof. Without being limited to any theory, these markers are associated with immune signaling, cell-cell and cell-ECM adhesion, targeting, pattern recognition, T-lymphocyte inhibition, increase in growth factor receptors, and inactivation. of pro-inflammatory proteins.

In particular, in one embodiment, a substantially homogeneous population of ASC is one where at least about 80%, such as at least about 85%, express CD10, CD140b, CD160, CD204, CD272, CD44, CD49a, CD54, CD9, Galectin 3, Galectin 9, HLA-G and / or LTpR, and optionally HLA-ABC, such as at least about 90% or in some cases at least about 95% or more express CD10, CD140b, CD160 , CD204, CD272, CD44, CD54, CD9, Galectin 3, Galectin 9, HLA-G and LTpR. In some embodiments, ASCs can be further characterized as expressing no more than about 20%, such as no more than about 15%, or in some cases no more than about 10%, of CD152, CD274, and / or CD86; and / or optionally, at least about 70% CD258, at least about 55% CD270, at least about 80% CD49a, up to about 30% CXCR4, and / or between about 5% and about a 35% of CD200. In some embodiments, the ASC population can also be one where, at most, about 15% of the ASCs express CD15, CD152, CD163, CD18, CD274, CD39, CD40, CD62L, CD80, CD86, and optionally HLA. -DR, -DQ, -DP.

In one embodiment, a substantially homogeneous population of ASC is one where at least 90% express CD10, CD140b, CD160, CD204, CD272, CD44, CD54, CD9, Galectin 3, Galectin 9, HLA-G, and LTpR; at least 80% express CD49a; at least 60% express CD258 and CD270 and at least 5% express CD200; at most 15% express CD15, CD152, CD163, CD18, CD274, CD39, CD40, CD62L, CD80 and CD86; and a maximum of 30% expresses CXCR4.

In one embodiment, a substantially homogeneous ASC population is one where at least 95% of the ASC population expresses CD10, CD140b, CD160, CD204, CD272, CD44, CD54, CD9, Galectin 3, Galectin 9, HLA-G and LTpR; at least 85% express CD49a, at least 65% express CD258 and CD270 and at least 10% of the population express CD200, and at most 15% of the population express CD15, CD152, CD163, CD18, CD274, CD39, CD40, CD62L, CD80, CD86, and HLA-DR, -DQ, and -DP, and at most 25% express CXCR4.

In one embodiment, the ASCs are further characterized by less than 20%, such as less than about 15%, such as less than about 10% of the CD274-expressing ASCs. Optionally, ASCs are also characterized by at least about 90%, such as at least about 95%, such as at least about 98% of CD54-expressing ASCs. In another specific embodiment, the ASCs are characterized by each marker in Table 15 in Example 10 at a population percentage in the range of the minimum value to the maximum value shown in Table 15.

ASCs can also be characterized by their stromal / stem cell marker expression percentages in accordance with one embodiment herein and their immunomodulation marker expression percentages in accordance with an embodiment herein. As a non-limiting example, a substantially homogeneous ASC population is one where

- at least 90% express CD90, CD73, CD13, CD29 and CD166; a maximum of 5% expresses CD45, CD19, CD14 and CD31; a maximum of 10% expresses CD106; between 2 and 15% express CD36; at least 10% express CD146; at least 80% express CD105 and at most 40% express CD34; Y

- at least 90% express CD10, CD140b, CD160, CD204, CD272, CD44, CD54, CD9, Galectin 3, Galectin 9, HLA-G and LTpR; at least 80% express CD49a; at least 60% express CD258 and CD270 and at least 5% express CD200; at most 15% express CD15, CD152, CD163, CD18, CD274, CD39, CD40, CD62L, CD80 and CD86; and a maximum of 30% expresses CXCR4.

In a further embodiment, ASCs are also or alternatively characterized by a change in one or more cell surface markers in response to a pro-inflammatory cytokine such as interferon-gamma. This can be advantageously tested according to the test of Example 11, typically by measuring a change in one or more ASC markers in Tables 16 and 17 which show

- a positive or negative change in the percentage of the ASC population that expresses the marker in at least 5% of the ASC population, or

- a positive or negative change in the level of expression of the marker in the portion of cells that express the marker by at least 0.5 times,

when cultured for 3 days in the presence of 50 ng / ml IFN-gamma, compared to a control, such as cells from the same ASCs that have not been stimulated with IFN-gamma.

For example, in some embodiments, upon stimulation with IFN-gamma, the percentages of the a Sc population expressing CD200, CD270, CD9, CXCR4 are reduced; the percentages of the ASC population expressing CD274 and CD49a increase, and the level of CD54 expression in CD54-positive cells increases.

In some embodiments, the change is one or more or all of

- the percentage of the ASC population that expresses CD274, CD106 and / or CD49a increases by at least 5%, such as the percentage of CD274 increases by at least 40%, such as at least 60%;

- the percentage of the ASC population that expresses CD200, CD270, CD9 and / or CXCR4 is reduced by at least 5%, such as the percentage of the ASC population that expresses is reduced by at least 10%;

- the level of expression of CD10, CD54, HLA-ABC and / or HLA-DR / DQ / DP increases in cells positive for markers, such as the level of expression of CD54 in cells expressing CD54 increases at least 20 times, such such as at least 35 times, such as at least 30 times; me

- the level of expression of LTpR decreases, for example, by at least 2 times.

In a specific embodiment, at most about 30%, such as at most about 20%, such as at most about 15%, such as at most about 10% of the ASC population expresses CD274, whereas with the stimulation with interferon gamma, at least about 70%, such as at least about 80%, such as at least about 85%, such as at least about 90%, such as at least about 95% of the population of ASC expresses CD274, for example, when ASCs are cultured for 3 days in the absence and presence of 50 ng / ml IFN-gamma, respectively.

Also notable is an increase in MFI from 3 to 97 for marker CD54 (ICAM-1) illustrating the mobilization of an intercellular adhesion molecule necessary for stabilization of ASC-leukocyte interactions and signal transduction. ICAM-1 is a ligand for LFA-1 (integrin), a receptor found on leukocytes. Thus, in one embodiment, at least 95% of the ASC population expresses CD54 and, upon stimulation with interferon gamma, the level of CD54 expression in CD54-expressing cells increases at least 20-fold, such as at least 30 times.

In a particular embodiment, after stimulation with interferon gamma, the percentage of the ASC population that CD274 expression is increased by at least 80% and the expression level of CD54 in CD54-positive cells is increased by at least 25-fold.

Compositions:

The present invention also provides compositions of each immunosuppressive substantially homogeneous adult human ASC population detailed in the aspects and embodiments herein, including those in the "ASC" section above and those obtained by each process in the "Process" section, in a protein-free cryoprotectant at a concentration of at least 1.5 x 107 cells per ml. Each of said ASC populations is also enabled in the format of a composition of the invention.

In particular, it has been found that ASCs, particularly ASCs obtained according to the process of the invention, can be cryopreserved at high concentrations in a protein-free cryoprotectant; at least about 1x107 cells per ml, without compromising viability, proliferation ability, immunosuppressive properties, or recovery. It has also been found that the CryoStor-based cryoprotectant can provide greater proliferation capacity than a human serum albumin (HSA) -based cryoprotectant.

In particular, the compositions of the invention may comprise a suspension of a substantially homogeneous population of adult human stem cells, isolated from adipose tissue collected from a donor, in a protein-free cryoprotectant, wherein the cell concentration is at least 1, 5 x 107 cells, such as at least 2 x 107 cells, such as at least 3 x 107 cells, such as at least 5 x 107 cells, per ml of added cryoprotectant. Preferably, the cell concentration is at least 2x107 cells, such as about 2.2x107 cells per ml of added cryoprotectant.

As described above, the protein-free cryoprotectant typically comprises from about 5% to about 15% DMSO and Trolox, Na +, K +, Ca2 +, Mg2 + 1Cl-, H2PO4-, HEPES, lactobionate, sucrose, mannitol , glucose, dextran-40, adenosine and glutathione. Preferably, the cryoprotectant comprises about 10% DMSO. Other protein-free cryoprotectants known in the art can also be used. Those already described in the "Process" section are particularly contemplated.

Compositions obtained are also enabled when frozen ASC compositions are thawed. Frozen ASC compositions can, for example, be thawed in a 37 ° C water bath or thawed / stored at room temperature in the operating room. Compositions where, immediately after thawing

(a) at least 85% of the ASC population are viable cells, and the viability after storage at room temperature for 2 hours is at least 80%;

(b) the ASC population has a proliferation capacity that provides a PD of at least 1 when cultured for 48 hours;

(c) the ASC population is capable of suppressing dendritic cell maturation and activation;

(d) recovery after thawing is greater than 95%, and recovery of cells after storage at room temperature for 2 hours after thawing is at least 85%

(e) the ASC population has in vitro cell adhesion such that at least 60%, such as at least 65%, such as at least 70% of the total number of cells are adherent after 5 hours in culture.

Compositions are also enabled, where, after storage at room temperature for 2 hours after thawing,

(a) at least 80% of the stem cell population are viable cells;

(b) the stem cell population has a PD of at least 1 when cultured for 48 hours;

(c) the stem cell population is capable of suppressing dendritic cell maturation and activation;

(d) the recovery is at least 85%, and

(e) the ASC population has in vitro cell adhesion such that at least 60%, such as at least 70% of the total number of cells are adherent after 5 hours in culture.

The present invention also provides pharmaceutical compositions. The compositions of the invention are sterile and free of endotoxins and mycoplasmas, the levels of endotoxin are normally determined with an analytical method that provides a minimum detection level of 10 EI per ml. Since the compositions of the invention are ready for clinical use, the pharmaceutical compositions normally comprise only the ASCs and the protein-free cryoprotectant. However, additional components are also contemplated. Specifically, the pharmaceutical composition may further comprise a soluble biomaterial or hydrogel containing natural or synthetic biopolymers such as extracellular matrix proteins, peptides or mucopolysaccharides and / or alginate. For example, the pharmaceutical composition may comprise sterile, endotoxin-free alginate (sodium alginate VLVG, Novamatrix, FMC Biopolymers, Norway), calcium partially cross-linked with D-gluconic acid and salt of hemicalcium (Follín et al., 2015). In one embodiment, the alginate is mixed with the ASCs and cryoprotectant to a final concentration of 1% (w / v) partially cross-linked alginate prior to the final cryopreservation step. In another embodiment, the partially cross-linked alginate is stored at RT and mixed with the final product at a final 1% (w / v) alginate concentration, for example, by injecting the ASC preparation into the alginate container. before aspirating the final suspension and connecting to the injection catheter.

Syringes or other means for injection or infusion of the ASC compositions, which contain the ASC compositions, are also provided. Typically, the ampoule containing the ASC composition is sterilized with an alcohol swab (82% ethanol and 0.5% chlorhexidine, Mediq, Denmark) and the cell suspension is aspirated with a needle into a sterile syringe. The syringe is then connected to an injection catheter, eg, a MYOSTAR injection catheter (Biological Delivery System, Cordis, Johnson & Johnson, USA) for injection. Injection within 3 hours after thawing is recommended. Preferably, the syringe or other means for injection or infusion contains about 5 ml of the composition, comprising from about 100 million to about 120 million cells, such as about 110 million ASC.

Therapeutic use:

In one embodiment, an ASC composition according to the invention is provided for use as a medicament, eg, for tissue regeneration, immunosuppression, and / or as an anti-inflammatory drug.

In fact, the invention provides various therapeutic uses for the ASC compositions of the invention, which are "ready to use" and ready for clinical use. Due to their high ASC concentration, at least 1.5 x 107 cells, preferably at least 2 x 107 cells, such as approximately 2.2 x 107 cells, per ml of cryoprotectant, the compositions can be used for applications where a small Injection volume is essential as well as for applications where high concentration compositions are diluted prior to administration and administered, for example, by infusion. Furthermore, ASCs from several different donors can be stored in a cell bank, allowing for repeated and / or more versatile treatment options.

Without being limited to any theory, after administration, ASCs stimulate and enhance regeneration through paracrine mechanisms that release extracellular substances that promote natural endogenous repair mechanisms, including matrix remodeling, revascularization, and immune modulation. Another inherent part of the immune modulation of ASC is active immunosuppression that prevents immunogenicity and thus rejection of the allogeneic ASC graft. This may be an innate feature of ASC that distinguishes these cells from other somatic cells.

Thus, in one embodiment, an allogeneic therapeutic is enabled for use in tissue regeneration, eg, in the treatment or prevention of an ischemic tissue disorder or other tissue dysfunction or destruction disorder. Non-limiting examples of ischemic tissue disorders include ischemic heart disease (with and without heart failure), acute myocardial infarction, ischemic stroke, critical limb ischemia, ischemic injury, and ischemic reperfusion injury / primary organ graft dysfunction. Non-limiting examples of tissue destruction or dysfunction disorders include intervertebral disc repair, non-ischemic dilated cardiomyopathy, and articular cartilage disorders. In some embodiments, from about 5x107 to about 5x108 cells in at most about 5 ml of a composition according to the invention are administered directly to ischemic tissue. In a specific embodiment, from about 5x107 to about 5x108 cells in a maximum of about 5 ml of a composition according to the invention are administered by intramyocardial injection to a patient with reduced left ventricular ejection fraction (EF) and insufficiency. cardiac. For example, a CSCC_ASC product comprising approximately 110 million cells in 5 ml of cryoprotectant can be thawed and administered in max 1, max 2, or max 3 hours by direct intramyocardial injection in a patient with reduced left ventricular EF and failure cardiac. In another specific embodiment, from about 5x107 to about 5x108 cells in a maximum of about 1 to about 5 ml of a composition according to the invention are administered by direct injection into a joint of a patient, for example, which have an articular cartilage disorder.

In one embodiment, an allogeneic therapeutic is enabled for use as an immunosuppressant, eg, to treat or prevent an autoimmune disease or disorder or transplant rejection. Non-limiting examples of autoimmune diseases and disorders include Crohn's disease, multiple sclerosis, type 1 diabetes, kidney disease, rheumatic arthritis, and rejection of a transplanted organ, including, but not limited to, bone marrow, heart, lung, and kidney transplants. In a specific embodiment, about 10x106 to about 0.5x106 cells per ml in about 200 ml of sterile infusion fluid of a composition according to the invention can be administered by intravenous or intraarterial injection or by infusion into a patient, for example, a patient suffering from Crohn's disease.

In one embodiment, an allogeneic therapeutic is enabled for use in treating or preventing inflammation, ie, as an anti-inflammatory drug. Non-limiting examples of inflammatory disorders include type 2 diabetes, kidney disease, non-ischemic dilated cardiomyopathy, arteriopulmonary hypertension, and sepsis. In In a specific embodiment, about 10x106 to about 0.5x106 cells per ml in about 200 ml of sterile infusion fluid of a composition according to the invention can be administered by intravenous or intraarterial injection or by infusion to a patient , for example, a patient suffering from arteriopulmonary hypertension.

For example, a CSCC_ASC product can be thawed, optionally diluted in 5 to about 200 ml of sterile infusion fluid at a concentration of about 10 x 106 to about 0.5 x 106 cells per ml, eg, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 million cells per ml; and administered in a maximum of 1, a maximum of 2 or a maximum of 3 hours by injection or infusion in a patient suffering from or at risk of an autoimmune or inflammatory disorder or transplant rejection. Alternatively, a CSCC_ASC product can be thawed and diluted in a sterile liquid to a concentration of approximately 12, 13, 14, 15, 16, 17, 18, 19, or 20 million cells per ml, and administered by intravenous or intra-arterial injection. or infusion, or administered directly into diseased tissue.

In some embodiments, multiple ASC compositions according to the invention can provide personalized treatment, for example by allowing tissue pairing between donor and recipient prior to treatment, various recipient treatments and, in case the recipient needs various treatments, the possibility of changing ASC from one donor to another. The latter is particularly useful in case the recipient has developed an alloantibody response to ASC from a previously administered ASC preparation, in which case it may not be possible to continue using ASC from the same donor. Specifically, a multi-donor ASC cell bank enables recipient-donor tissue pairing, which can improve clinical efficacy.

The present invention is illustrated by the following Examples, which are not intended to be limiting.

Example 1

ASC Product Manufacturing

The following procedure has been used:

Isolation of the vascular fraction of the stroma:

The liposuction is obtained from healthy donors. Donor eligibility is determined based on a donor interview, questionnaire, and tests for infectious disease markers HIV, hepatitis B and C, syphilis, and HTLV. Subcutaneous abdominal fat liposuction is performed under local anesthesia and provides approximately 100 ml to 300 ml of liposuction from each donor. Local anesthesia is placed on the abdominal skin, in 2-4 places for the subsequent connection of liposuction. Through these holes, the infusion cannula (thin needle) is inserted while injecting liquid, for example, anesthetics and reagents to relax fatty tissue, such as a lactate buffer, sodium bicarbonate, adrenaline, and lidocaine. Bodyjet EVO (water assisted liposuction) (Human med, Germany) is used. The liposuction is withdrawn through the Bodyjet suction cannula and into the sterile collection chamber. The liposuction is transferred to a sterile flask / bottle with a sterile syringe.

The stromal vascular fraction is isolated from the lipoaspirate by enzymatic digestion of adipose tissue before expansion of the culture in bioreactors. The liposuction is washed twice with phosphate buffered saline (PBS) pH 7.4 to remove residual blood. The adipose tissue is digested by incubation with 0.6 PZ U / ml of collagenase n B6 (Serva GmbH, Germany) dissolved in HBSS (+ CaCl2 MgCl2) (Gibco, Life Technologies) diluted to a concentration of Ca2 + 2 mM at 37 ° C for 45 minutes in constant rotation. Collagenase is neutralized with medium containing 5% human platelet lysate and 1% penicillin / streptomycin and filtered through a 100 µm filter (Steriflip, Millipore). The remaining cells are centrifuged at 1200 g for 10 minutes at room temperature, resuspended and counted using a NucleoCounter® NC-100TM according to the manufacturer's instructions.

First step expansion:

Approximately 100 million cells of the SVF are loaded into a bioreactor (Quantum Cell Expansion System, Terumo, Belgium) for ASC expansion. The quantum bioreactor is a functionally closed system consisting of a disposable hollow fiber bioreactor enclosed in a separate incubator. It is fed through two circulation circuits with inlets for media and reagents or cells. Waste is disposed of in a waste bag.

The entire process is computerized and controlled by a touch screen interface, allowing control of the mean perfusion rate, collection time, media washes, and other tasks associated with ASC growth. With the exception of filling inlet bags with cryoprecipitate; the addition of enzymes for cell detachment; or transferring harvested cells from the collection bag to the cell inlet bag (all of which is done prior to loading into the bioreactor), all procedures associated with the bioreactor are closed or protected by a sterile 0.2 mm barrier filter.

Four to 24 hours before loading the SVF into a bioreactor, the system is primed with phosphate buffered saline (PBS) and subsequently loaded with approximately 30 ml of cryoprecipitate (Blood Bank, Rigshospitalet, Denmark) for coating of the bioreactor. The cryoprecipitate is used as is or diluted, for example, 1: 3 or 1: 4, such as to a total of 100 ml, with PBS. Before loading the cells, the PBS and cryoprecipitate are washed from the system and replaced with complete medium (Minimal Essential Medium, alpha MEM (aMEM) without ribonucleosides and deoxyribonucleosides, (Gibco, Life Technologies), 1% penicillin / streptomycin ( Gibco, Life Technologies), 5% human platelet lysate (Stemulate, Cook General Biotechnology) Gas is supplied to the system as a premix supply of 20% O2, 5% CO2, and equilibrated with N2 (Strandm0llen, Denmark) .

100 million SVFs diluted in 100 ml complete medium are transferred to a cell inlet bag using a 60 ml syringe and loaded into the quantum bioreactor. Cells are allowed to adhere for 24 h, after which continuous media feeding is activated. The medium feed rate starts at 0.1 ml / min. Based on glucose and lactate measurements, the rate of feeding is adjusted and cell expansion is identified. Glucose and lactate levels are monitored by removing samples from the quantum bioreactor sample port and taking measurements on an ABL 835 FLEX blood gas analyzer (Radiometer, Denmark). Approximately 9 days after cell loading, ASCs are harvested by loading a TrypLE® Select (Gibco, Life Technologies) into the system. The harvesting process includes a wash of the system with PBS, the addition of 180 ml of TrypLE® Select and a 20 minute incubation. The collected cells are washed in the cell collection bag. The cells from the collection bag are transferred to centrifuge tubes in laminar air flow, washed, granulated, and counted and tested for viability with a NucleoCounter.

Intermediate ASCs from the first pass are pelleted and resuspended in CryoStor10 (BioLifeSolutions) at a concentration of 10 million cells per ml. This solution is aliquoted into CellSeal cryogenic vials (Cook General Biotechnology) in a total volume of 5 ml per vial.

Cryogenic vials are then frozen in a speed controlled freezer (Kryo 560-16, Planer) to reach -80 ° C and transferred on dry ice to a liquid nitrogen dry storage isothermal liquid nitrogen freezer (V1500-AB , CBS) that provides uniform temperatures in the -180 ° C range, with no liquid nitrogen in the sample storage space. This system minimizes the risk of cross contamination.

Cryopreserved first pass intermediates make up the work cell bank and are stored until loading for the second expansion in the bioreactor.

Second step expansion

Four to 24 hours prior to loading the intermediate ASCs from the first pass for the second pass expansion, a new bioreactor is primed and coated as described for the first pass expansion. The system is charged with complete medium and gas is supplied to the system as a pre-mixed supply of 20% O2, 5% CO2, and equilibrated with N2. A CellSeal vial containing 50 million ASC is withdrawn from the first pass of the nitrogen tank. The cell ampoule is transferred to a "resealable bag" and thawed in a 37 ° C water bath. The seal at the bottom of the vial is removed and disinfected with alcohol. A piece of breather tube is cut, to avoid too much pressure on the cells. Using a 10 ml syringe and a 16G needle, a predetermined number of cells (for example, 1 quarter of the ampoule, 1 half of the ampoule, or the entire ampoule) are withdrawn and transferred to a 10 ml centrifuge tube. . Cells are counted and viability is determined with a NucleoCounter. Cells are diluted with 100 ml of complete medium and transferred to the cell inlet using a 60 ml syringe. Expansion and harvest are performed as described for the expansion of the first step.

After approximately 7 days of second pass expansion, ASCs are harvested, pelleted, and cells are resuspended in CryoStor10 at a concentration of approximately 22 million cells per ml, typically in the range of approximately 20 to approximately 24 million cells. per ml. This solution is aliquoted into CellSeal cryovials in a total volume of 5 ml per vial, containing approximately 110 million cells per vial, typically in the range of about 100 to about 120 million cells per vial.

The cryovials are then frozen in a speed-controlled freezer to reach -80 ° C and transferred on dry ice to a liquid nitrogen dry storage container providing uniform temperatures in the -180 ° C range, with no liquid nitrogen in the storage space of the sample. The cryostored second-step ASCs constitute the investigational drug CSCC_ASC and are stored in the product's cell bank until shipment for clinical use.

The product is ready to use and requires only minimal handling with the patient. A frozen vial is thawed in a 37 ° C water bath in / near the operating room. The vial is sterilized (usually with a cotton swab alcohol) and the cell suspension is aspirated with a needle into a sterile syringe. The syringe is then connected to the injection catheter and administered to the patient.

Example 2

Characterization of cell surface markers of the ASC product

Immunophenotyping was used to identify cell quality and was performed on the intermediate of the first step and the final product of the second step before and after cryopreservation for the ASC product prepared according to Example 1.

The collected cells were washed, filtered and distributed to tubes with or without antibodies. Cells were incubated for 30 minutes at room temperature with the antibodies shown in Table 1. After incubation, cells were washed, centrifuged, and resuspended in PBS for flow cytometry using a six-color protocol.

According to the European Pharmacopoeia, flow cytometry is a suitable method for the identification of cell surface markers. Fluorescence-labeled antibodies against the surface markers recommended by ISCT / IFATS (International Federation of Adipose Therapeutics and Science and the International Society for Cell Therapy) for identification of ASCs were added to ASCs and cell-associated fluorescence was measured. using a flow cytometer. For the characterization of a given product, a 6-color compensated protocol is used that labels cells with fluorophores phycoerythrin, fluorescein isotyanate, phycoerythrin-Texas Red, phycoerythrin-cyanine and allophycocyanin. Viability was determined by SYTOX blue staining. The protocol was developed with manual compensation, isotypic controls, and Fluorescence Minus One controls. Dead cells and doublets were excluded from the final analysis. Data was collected and analyzed using a GMP compatible Navios (Beckman Coulter, Germany). Data were analyzed using Navios and Kaluza software (Beckman Coulter, Germany).

TABLE 1

TABLE 2

continuation

TABLE 3

Example 3

ASC product characterization - cell viability

Product quality and final product effect are subject to ASC feasibility. Therefore, viability is significant throughout the manufacturing process.

Viability was determined several times during the production process of Example 1; SVF viability was determined before loading into the bioreactor for the first expansion; the viability of expanded ASC from the first step was determined after harvesting and after thawing and loading in the second step; the viability of the final product was determined after harvesting and after cryopreservation and thawing. The Percentage viability was determined with a NucleoCounter® NC-100 ™. The Nucleo-Counter is an imaging cytometer based on the fluorescence detection of the DNA-binding fluorescent dye, propidium iodide (PI).

TABLE 4

Example 4

Differentiation tests

The osteogenic, adipogenic and chondrogenic differentiation capacity of the second-pass ASCs expanded with Stemulate as a growth supplement in quantum bioreactors was determined using the StemPro differentiation kit (Gibco, Life Technology), according to the manufacturer's protocols.

For osteogenic differentiation, 10,000 ASC / well were incubated in 12-well plates in osteogenic induction medium (StemPro basal osteocyte / chondrocyte differentiation medium, StemPro osteogenesis complement, penicillin / streptomycin). For adipogenic differentiation, 20,000 ASCs / well were incubated in 12-well plates in adipogenic induction medium (StemPro basal adipocyte differentiation medium, StemPro adipocyte complement, penicillin / streptomycin). For chondrogenic differentiation, multiple 5 µl drops of 80,000 ASC were incubated in chondrogenic induction medium (StemPro basal osteocyte / chondrocyte differentiation medium, StemPro chondrogenesis complement, penicillin / streptomycin). Cells were induced for 21 days, with the medium changed every 3-4 days. Control cells were incubated with complete medium without complement until confluence.

Osteogenic differentiation was documented as cells showed calcium deposits with Alizarin Red S staining (Sigma-Aldrich). Adipogenic differentiation was documented through the morphological appearance of the fat droplets stained with Oil Red O (Sigma-Aldrich). Chondrogenic differentiation was documented as cells were stained with Alcian Blue 8GX (Sigma-Aldrich). Control cells kept in complete media were all negative.

Example 5

Comparison of cryoprotective formulations

The viability and function of the ASCs in the final cryopreserved product when thawed together with the patient is of primary importance to the efficacy of the product. Viability and function were determined for cryopreserved ASCs with the use of different cryoprotective formulations.

The intermediate ASC products from the first step and the final ASC products from the second step were manufactured as described in Example 1. The intermediate ASCs were frozen in different cryoprotective formulations at 50 x 106 cells per 5 ml in cryogenic vials.

The final ASCs of the second step were frozen in different cryogenic formulations at 100 x 10 6 cells per 5 ml in cryogenic vials. Cells were frozen in an automatic freezer at -80 ° C, stored in dry liquid nitrogen storage, and thawed in a 37 ° C water bath; viability and recovery were determined immediately after thawing and up to 3 hours after thawing while kept in cryogenic formulation at room temperature.

Immediately after thawing and up to 3 hours after thawing, while kept in cryogenic formulation at room temperature, the final ASC cell product was washed and cultured for identification of cell function as determined by morphology, adherence and proliferation in vitro. The cultures were established with 1x10 6 cells per T75 flask with 20 ml of complete medium, 37 ° C, 5% CO2.

Viability and recovery were determined with a Nucleocounter. Morphology and adhesion were determined by microscopy 24 h after the cells were cultured. Proliferation was determined by detaching and counting cells with a Nucleocounter 48 hours after they were cultured.

Cryoformulations containing 5% or 10% HPL or human albumin (HA) were analyzed. English) and DMSO in isotonic saline, CryoStor10 and CryoStor5.

TABLE 5

TABLE 6

TABLE 7

Cell function and potency are the best indicators of clinical efficacy. With currently available in vitro methods, morphology, adhesion, and proliferation are the best general indicators of cell function. Microscopic examination revealed that cell function (morphology, adhesion, and proliferation) was superior for cells formulated in CryoStor10.

TABLE 8

TABLE 9

TABLE 10

Example 6

Growth plug-in comparison

SVF was isolated from liposuction obtained from three healthy female donors (age 32-47 years; mean age 40 years) as described in Example 1. SVF was cultured in four different GMP-compatible media containing 5% hPL or 10% FBS. ASC P0, P1 and P5 were characterized and used for the analysis.

Primary ASC cell cultures were established by seeding 4.5 x 106 SVF / T75 flasks in complete medium containing Minimal Essential Medium, MEM Alpha (aMEM), 1% Penicillin / Streptomycin and with four different growth supplements:

1. 5% Human Platelet Lysate (PLTMax, Mill Creek Life Sciences), 10 IU Heparin

2. 5% human platelet lysate (Stemulate, hPL-S, COOK General Biotechnology), 10 IU heparin 3. 5% human platelet lysate (Stemulate, hPL-SP, COOK General Biotechnology)

4. 10% Fetal Bovine Serum (FBS, Gibco, Life Technologies)

HPL is composed of plasma with fibrinogen and other clotting factors, therefore heparin must be added to avoid gelatinization. COOK General Biotechnology produces StemulateTM Pooled Human Platelet Lysate in two different versions, PL-S which requires heparin and PL-SP which does not require heparin, where some of the clotting factors have been removed and the addition of heparin is not required.

Cells were incubated under standard conditions at 37 ° C in humid air with 5% CO2. The ASC medium was changed after 2 days to discard non-adherent cells, and every 3-4 days thereafter. When the culture reached a confluence level of approximately 90%, the cells were washed with PBS, separated, and passaged for experimental setups. Proliferation was determined on days 1, 2, 3, 5, and 7 by manual counting in a Bürker-Türk chamber. Population doublings (PD) were calculated as PD = In (N / N0) / In 2, where N is the number of cells harvested on day 7 and N0 is the number of cells seeded. The osteogenic, adipogenic and chondrogenic differentiation capacity of ASCs was determined using the StemPro differentiation kit. Immunophenotyping was determined as described in Example 2. Genomic stability was determined by comparative genomic hybridization.

TABLE 11

As illustrated in Table 11, the use of an HLP product is clearly a more efficient growth supplement in terms of proliferative capacity than the use of FBS.

ASCs grown with all growth supplements tested in step one were shown to have profiles immunophenotypic comparable and characteristic of ASC and maintained their ability to differentiate from three lineages. Genomic stability identified by Comparative Genomic Hybridization (CGH) shows that in vitro expanded ASCs, in the presence of PLTMax, Stemulate, and FBS did not show any unbalanced chromosomal rearrangement when cultured up to a fifth passage. Therefore, the higher proliferative capacity of ASCs grown in PLTMax or Stemulate did not compromise genomic stability.

Example 7

Comparison of manual and automated expansion in bioreactors

SVF was isolated from abdominal fat, suspended in basal medium supplemented with penicillin / streptomycin and a growth supplement containing serum, and seeded in T75 flasks or in a quantum bioreactor that had been coated with cryoprecipitate. The cultivation of ASC passed from SVF was carried out through three methods: flask to flask; flask to bioreactor; and bioreactor to bioreactor. In all cases, quality controls were performed by sterility, mycoplasma, and endotoxin tests, in addition to evaluation of cell counts, viability, and immunophenotype as determined by flow cytometry.

Primary flask cultures were established by seeding 4.5 x 106 SVF cells per T75 flask incubated under standard conditions at 37 ° C, 5% CO2. The culture medium was changed after 3 days removing non-adherent cells. Subsequently, the medium was changed every 3-4 days for the rest of the culture. Reaching a confluence level of 90%, cells were harvested. Cells were reseeded at 3.5 x 105 cells / T75 flask. Viability and yield of ASCs in step 0 (P0) and step 1 (P1) were determined with a NucleoCounter® NC-100TM and calculated as averages of three t 75 flasks.

For primary expansion of SVF in the quantum bioreactor, the system was primed and coated with cryoprecipitate as described in Example 1. 100 x 106 SVF cells were loaded and allowed to bind for 24 hours, before feeding to 0.1 ml / min will start automatically. After 3 days of culture, a washing task was performed to remove non-adherent cells. For second-pass expansion of previously cultured ASCs (ie, those resulting from the primary expansion), the quantum bioreactor was seeded with 30 x 106 ASCs. The SVF and ASC expansion performed in the quantum bioreactor used media and reagents that were identical to those used for flask culture.

TABLE 12

The viability of the P0 and P1 ASCs was greater than 96%, regardless of the culture in a flask or bioreactor. ASC expanded in all conditions were shown to have comparable and characteristic ASC immunophenotypic profiles. Sterility, mycoplasma, and endotoxin tests were consistently negative.

However, an average of 55 x 106 SVF cells loaded into a bioreactor produced 89 x 106 ASC P0 (1.6 times more ASC relative to the number of SVF cells seeded), while 4.5 x 106 SVF seeded per flask T75 produced an average of 1.75 x 106 ASC (0.4 times the number of ASCs relative to the number of SVF cells seeded). The expanded P1 ASCs in the quantum bioreactor demonstrated a population doubling (PD) of around 2.1 regardless of whether P0 was grown in flasks or in the quantum bioreactor, while the P1 ASCs in flasks only achieved a PD of 1, 0.

In conclusion, ASC fabrication in a bioreactor improves ASC expansion rate and performance significantly relative to manual T-flask processing, while maintaining the purity and quality essential for safe and consistent cell production.

Example 8

Comparison of growth complements in quantum bioreactors

The use of HPL (Stemulate, hPL-SP, heparin-free, COOK General Biotechnology) and fetal bovine serum (FBS) (Gibco, Life Technology) as growth supplements for ASC expansion in a quantum bioreactor were compared.

From liposuctions from three donors, SVF isolation from abdominal fat and first pass expansion were performed according to Example 1. Of these steps, 30 x 106 ASC were reloaded into a new quantum bioreactor for a second expansion. In all the steps, the metabolic control (glucose and lactate) guided the feeding speed and the collection time. The viability, sterility, purity, differentiation capacity and genomic stability of ASCs were determined. Microbial quality controls, flow cytometry, triple differentiation, and genomic stability were performed as determined by comparative genomic hybridization matrix assays.

Each step of this two-step process demonstrated that HPL supported ASC proliferation to a greater extent than FBS. As an average of 3 donors, culturing SVF in HPL media for 9 (7-11) days yielded an average of 546 x 106 a Sc. Culture of SVF in FBS media required an additional 8 days to produce 111 x 106 As C. Second pass ASCs produced on average 800 x 106 cells (PD 5.2) after 6 days in hPL media compared to 100 x 106 (PD: 2.2) cells in FBS media after 21 days. The ASCs met the ISCT criteria in both types of media (immunophenotype and triple differentiation). Comparative genomic hybridization demonstrated genomic stability. Sterility, mycoplasma, and endotoxin tests were all negative.

The combination of the use of quantum bioreactors and hPL, recovered 5 times more first pass ASC and 8 times more second pass ASC compared to the use of quantum bioreactors and FBS.

Example 9

Immunosuppressive activity of the final ASC product

A manifest during tissue damage of any origin, be it ischemic, traumatic or autoimmune in nature, is the rapid appearance of inflammatory cells, which slowly diminishes during the successful regeneration process but persists during the chronic healing of ischemic / traumatic wounds and the autoimmune reaction. This infiltrate consists of an initial accumulation of monocytes / macrophages followed by lymphocytes. As such, the immunosuppression exerted by an ASC product primarily in the monocyte / macrophage population is expected to shift the balance in favor of regeneration. The immunosuppressive activity of ASC is also imperative for allograft survival and thus a multitude of regenerative mechanisms.

As immunosuppression is an inherent ASC feature of interest for both allogeneic use and efficacy, in vitro cell models addressing innate and cellular immunity, such as human dendritic cell assays and human mixed lymphocyte reactions, were used to examine immunosuppressive activity.

Co-cultures with ASC and dendritic cells (DC) from circulating monocytes were established according to the method of Jensen and Gad (2010). Peripheral blood mononuclear cells were isolated from buffy coats of healthy donors younger than 50 years, by centrifugation on a Ficoll-Paque gradient (GE Healthcare) and isolation of the cell interface. Cells were washed in RPMI 1640 medium (Roswell Park Memorial Institute) (Sigma) with 1% pen / strep, and CD14 + monocytes were isolated by positive selection using a MACS sorting column and magnetic beads (CD14 MACS microbeads, human, Miltenyi Biotec). The column was washed with degassed PBSE buffer (PBS, 0.5 mol / l EDTA, 2 FBS), before and after applying the cell suspension. CD14 + monocytes were seeded in six-well plates at a density of 2 x 106 cells / ml in RPMI 1640 medium with 1% pen / strep, 2% human serum AB, recombinant human granulocyte macrophage colony stimulating factor (20 ng / ml) and human interleukin (IL) -4 (20 ng / ml; PeproTech). The medium was changed every 2-3 days. After 6 days of differentiation, cells were harvested and prepared for DC: ASC co-culture. The cell product CSCC_ASC; the second step of cryopreserved ASCs (in CryoStor10) was thawed and cells were placed in a culture flask (aMEM, 5% Stemulate, 1% pen / strep) for one week before starting the DC: ASC co-culture (ASC rehabilitated in cultivation). On the day of the DC: ASC pooled culture, one vial of CSCC_ASC cell product; cryopreserved ASCs from the second step (in CryoStor10) were thawed for direct use in pooled cultures. ASCs were seeded in a 48-well plate at a concentration of 1x106 per well, resulting in a 1: 1 DC to ASC ratio. DCs were activated by stimulation with 1 pg / ml lipopolysaccharide (LPS; Sigma) and 20 ng / ml interferon gamma (IFN-g; Peprotech). As a positive control for DC activation, a well containing DC alone was also stimulated with 1 pg / ml of lipopolysaccharide (LPS; Sigma) and 20 ng / ml of interferon gamma (IFN-g; Peprotech) and incubated for 24 hours.

Flow cytometry of dendritic cell maturation markers

The pooled cultures were harvested by resuspending and removing the supernatant and scraping the bottom. of the wells with a bent pipette tip. Harvested cells were washed twice with FACS wash buffer (PBS supplemented with 1.5% NaN3 and 1% heat inactivated FBS). Samples were incubated with human IgG (Sigma) on ice for 15 minutes to block Fc receptors. The primary antibodies used were IgG-APC, CD11c-APC, IgG1k-PE, CD40-PE, CD80-PE, CD86-PE (BD Bioscience) and HLA-DR-FITC (Beckman Coulter). Samples were incubated with antibodies for 30 minutes on ice and washed, and data was obtained on an Accuri FACS (BD Bioscience). Data were acquired in controlled events such as the DC population by CD11c positivity and size and analyzed in Flowlogic. Mean fluorescent intensity (MFI) was measured and compared to the MFI of positive control DCs.

TABLE 13

Human dendritic cell assays showed that the final cryoformulated second-pass ASC product suppressed human dendritic cell maturation / activation. This designated that the cell product was actively immunosuppressive and would escape rejection after injection in vivo. Experiments were performed with the final product directly after thawing and with ASC of the final and thawed product that had been put into culture for rehabilitation. Therefore the influence of ASC formulation and cryogenic storage with respect to dendritic cell activation had been addressed.

The immunosuppressive activity of the final ASC product of the second step was further examined in an in vitro cell model based on a mixed lymphocyte reaction (MLR). In this model, circulating peripheral blood mononuclear cells (PBMC) were stimulated by irradiated PBMC from an allogeneic donor and different proportions of final ASC product were added. Briefly, ASCs were grown in 96-well plates in proportions corresponding to 1:20 to 1: 1 of the following responding PBMCs. Wells without ASC (medium only) were used as positive controls. The day after, peripheral blood mononuclear cells (PBMC) were purified from buffy coats by density gradient centrifugation using Lymphoprep. Half of each PBMC group was irradiated with a gamma radiation source (3000 RAD). The PBMCs were combined in 96-well plates (100 µl 100 µl per well) to make a series of unique MLRs; proliferative pools of PBMC were stimulated by irradiated pools from other donors. After a co-cultivation period of 4 days, 3H-thymidine (25 pSi / ml) was added and incubated for 18-20 hours. Using an automated harvester, cells were harvested on filter plates, placed in scintillation liquid, and counts per minute were determined, visualizing proliferative responses of PBMC, with a scintillation counter (Topcount).

The addition of the final ASC product from the second step to MLR demonstrated that ASCs exert suppressive effects on the rapid proliferation of lymphocytes that normally occurs.

TABLE 14

Example 10

Immunomodulatory phenotyping of the final product / ASC of the second step

Phenotypic characterization of the final CSCC_ASC product, prepared according to Example 1, based on immunomodulatory surface markers was performed by flow cytometry. ASCs were harvested with TrypleSelect, washed, filtered, and distributed to tubes with or without antibodies. Cells were incubated for 30 minutes at room temperature with the antibodies shown in Table 15. After incubation, cells were washed, centrifuged, and resuspended in PBS for flow cytometry.

A single color protocol was used that labels the cells with antibodies conjugated to fluorophores such as phycoerythrin, flourescein isotyanate, phycoerythrin-Texas Red, phycoerythrin-cyanine, allophycocyanin and Brilliant Violet (conductive polymers). Viability was determined by staining with FVS-780 (Becton Dickinson). Dead cells and doublets were excluded from the final analysis. Data was collected and analyzed using a GMP compliant Navios (Beckman Coulter, Germany). Data were analyzed using Navios and Kaluza software (Beckman Coulter, Germany).

TABLE 15

All the CD markers shown in Table 15 have been chosen for their relevance in immunomodulatory and especially immunosuppressive functions. The characterization of a Sc as shown in Table 15 agrees with considerable immunosuppressive potential (Krampera et al., 2013). Especially the strong positive markers CD10, CD140a, CD160, CD204, CD258, CD270, CD272, CD44, CD49a, CD54, CD9, Galectin 3 and Galectin 9, HLA-G and LTpR reflect immunomodulatory capabilities of ASC through mechanisms such as signaling. immune system, cell-cell and cell-ECM adhesion, targeting, pattern recognition, inhibition of T lymphocytes, increased growth factor receptors, and inactivation of pro-inflammatory proteins.

Example 11

Immunomodulatory functional capabilities; phenotypic adaptations to the pro-inflammatory environment

To examine whether the immunomodulatory and immunosuppressive phenotype illustrated in Example 10 meets the actual immunosuppressive responsiveness, the ASCs of the product CSCC_ASC, prepared according to Example 1, were challenged with a pro-inflammatory cytokine in vitro and phenotypic changes were examined. .

The ASCs of the final product from different donors were cultured in vitro in standard medium (aMEM, Pen / Estrep, hPL 5%). At approximately 80% confluence, half of the cultures from each donor were stimulated with 50 ng / ml IFN-gamma, 25 ml of medium per culture. After 3 days, the cultures were harvested with TrypleSelect and prepared for flow cytometry as described in Example 10.

TABLE 16

TABLE 17

continuation

As shown in Table 16 and 17, the ASCs of the final product are capable of responding to an inflammatory environment. The answers are found at the population level as well as at the individual cellular level.

Population percent means the percentage of cells in the entire population that show the mentioned surface marker. The numbers for the mean fluorescence intensity correspond to the increase or decrease in the number of markers in each individual cell.

Of particular importance is that an 87% increase in CD274 (PD-L1) based on population illustrates the ability to initiate immunosuppressive actions. CD274, known for its expression in macrophages, plays an important role in suppressing the immune system during particular events such as tissue allografts and autoimmune diseases (Camillieri et al, 2016; Krampera et al., 2013). PD-L1 binds to its receptor, PD-1, found on activated T lymphocytes, B lymphocytes, and myeloid cells, and modulates the activation or inhibits the proliferation of T lymphocytes. PD-L1 acts at least in part through the induction of apoptosis. The precise role of CD274 in second-pass ASC has not yet been determined, but CD274-positive bone marrow MSCs, manually expanded with FBS, regulate T-cell proliferation and Th17 polarization, and have shown comparable effects on IFNgamma licenses.

Also notable is an increase in MFI from 3 to 97 for marker CD54 (ICAM-1) illustrating the mobilization of an intercellular adhesion molecule necessary for stabilization of ASC-leukocyte interactions and signal transduction. ICAM-1 is a ligand for LFA-1 (integrin), a receptor found on leukocytes.

A significant increase in HLA DR / DP / DQ is expected, but in the context of a lack of increase in the known costimulatory markers CD40, CD80, and CD86, an antigen-presenting phenotype is not obtained. On the contrary, the simultaneous significant increase in CD274 and the persistent expression of other immunosuppressive markers is consistent with the net production being anti-inflammatory (Krampera et al., 2013; Galipeau et al., 2016)

The ability to respond phenotypically and functionally to the environment is critical to the ability of the products to interact with the in vivo environment into which cells are injected during treatment. This inherent interactive feature of the product is significant for its characteristics and clinical efficacy. The ability to assume an immunosuppressive phenotype allows and explains allogeneic use.

Example 12

Clinical study

10 patients aged 62.5 ± 6.6 years (mean ± SD) with chronic ischemic heart disease and heart failure, reduced left ventricular failure (<45%), New York heart failure (NYHA) Class II-IN, without further revascularization options and in maximum tolerable medical therapy, have been treated with the final product CSCC_ASC. With a NOGA Myostar® catheter (Biological Delivery System, Cordis, Johnson & Johnson, USA), approximately 15 injections of 0.3 ml of CSCC_ASC prepared according to Example 1 were injected into viable myocardium in the border area of the infarcted area.

The patients underwent echocardiography and contrast-enhanced computed tomography at the beginning of the study and after six months.

A 320 multiple detector CT scanner (Aquilion One, Toshiba Medical Systems Corporation, Otawara, Japan) was used to perform cardiac computed tomography. R-R interval and multisegmental image reconstruction were performed with the scanner software. Images were reconstructed with 0.5 mm slice thickness and 0.25 mm increments in a 2% interval in the prospective window.

Echocardiography measured cardiac function and volumes in parasternal and apical views.

All imaging data was analyzed with the cvi post-processing tool (Circular Cardiovascular Imaging, Calgary, Alberta, Canada). The endocardial and epicardial borders were traced manually in end diastole and end systole and the mitral plane was established to define the basal border of the LV.

The New York Heart Association (NYHA) Functional Classification was used. Patients are placed in one of four categories based on how limited they are during physical activity with respect to shortness of breath.

The 6-minute walk test (6MWT) was standardized according to the American Thoracic Society guidelines from March 2002 using a 30-m (100-foot) long hallway or corridor. The Borg CR10 scales were used to measure the intensity of the experience.

TABLE 18

Six months after treatment, left ventricular ejection fraction (LVEF) and 6MWT improved, while left ventricular end-systolic volume (LVESV) and NYHA score decreased . The measurements reveal a better pumping function of the heart, greater work capacity and fewer attacks of shortness of breath. The treatment was safe and effective.

Example 13

ASC surface adhesion

An inherent characteristic that defines a population of ASCs is the adhesion capacity of ASCs. Furthermore, the ability to adhere is the first of many traits that demonstrate that ASCs have maintained normal cellular function. In this example, the ability to adhere to ASCs of the product CSCC_ASC, produced as described in Example 1 after storage in liquid nitrogen dry storage vessels at -180 ° C for up to 12 months, was studied. Briefly, ASCs from three donors, prepared according to Example 1, were stored for 1, 3, 6, and 12 months, thawed, counted, and cultured in vitro, 1 million viable cells per flask in aMEM with 5% HPL for 5 hours. After 5 hours, the cultures were washed and the adherent population was removed with TrypleSelect and counted.

TABLE 19 A B C

continuation

The ability to adhere to a surface is an important characteristic of ASC. The CSCC_ASC product exhibits a high degree of ASC adhesiveness maintained after freezing, storing and thawing.

If stored in N2 dry storage containers at -180 ° C for up to 12 months, 71-89% (mean: 76%) of cells retain functional ability to adhere.

Example 14

Surface markers

Cells produced as described in Example 1 were characterized as described in Example 2, except for the use of a single color flow cytometry protocol, producing the results shown in Table 20 below for ASC from the first pass (8 batches) and ASC from the second pass (14 batches).

Briefly, cells were analyzed directly after harvest and before cryopreservation. For flow cytometry, cells were washed with PBS, adjusted to 1 million cells per ml, and stained with the FVS-780 viability stain (Becton Dickinson) for 10 minutes at RT in the dark. The FVS-780 was washed with PBS containing FBS, the cell suspension was filtered and the antibodies were added for 30 minutes at RT in the dark and in volumes according to the previous titration. All the antibodies used were from Becton Dickinson and were conjugated with fluorochromes PE (Phycoerythrin), Bright Violet 510, FITC (fluorescein isothiocyanate), APC (Allophycocyanin) and PerCp-Cy5.5 (peridinin and chlorophyll tandem). Dead cells and doublets were excluded from the final analysis. Data was collected and analyzed using a GMP compatible Navios (Beckman Coulter, Germany). Data were analyzed using Navios and Kaluza software (Beckman Coulter, Germany).

TABLE 20

Table 20 shows the percentages of ASC after the first and second pass expressing named surface markers used for the characterization of ASC. Mean expression values are based on 8 and 14 batches for the first step and the second step, respectively. The minimum and maximum expression levels are also shown.

REFERENCE LIST

Bourin P, et al. Cytotherapy. June 2013; 15 (6): 641-648.

Camilleri et al. Stem Cell Research & Therapy (2016) 7: 107

Dominici M, et al. Cytotherapy (2006) Vol. 8, No. 4, 313-317.

Ekblond A. Presentation at the International Congress on Adipose Stem Cell Treatments 2015 (iCAST2015). Follin B, et al. Cytotherapy. 2015 Aug; 17 (8): 1104-18).

Gebler A, et al. Trends in Molecular Medicine 18.2 (2012): 128-134.

Jensen SS and Gad M. J Inflamm (Lond) 2010; 7:37.

Krampera et al., Cytotherapy, 2013; 15: 1054-1061

Mathiasen AB, et al. Am Heart J. 2012 164 (3): 285-91.

Mathiasen AB, et al. Int J Cardiol. 2013 170 (2): 246-51.

Qayyum AA, et al. Regen Med. 2012 7 (3): 421-8.

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WO 2014/203267 A2 (Kaziak Research PVT Ltd.)

Document WO 2006/037649 A1 (Cellerix S.L. and Universidad Autónoma de Madrid)

WO 00/02572 A1 (Baust J.G.)

WO 2010/064054 A1 (Reneuron Ltd.)

Claims (21)

A composition comprising a suspension of a substantially homogeneous and immunosuppressive population of adult human adipose tissue (ASC) stem cells in a protein-free cryoprotectant at a concentration of at least 1.5 x 107 cells per ml. The composition of claim 1, wherein at least about 80% of the ASC population expresses CD90, CD73, CD13, CD105, CD29, CD166, CD10, CD140b, CD160, CD204, CD272, CD44, CD49a, CD54 , c D9, Galectin 3, Galectin 9, HLA-G and LTpR and at most approximately 15% of the ASC population expresses CD45, CD19, CD14, CD106, CD31 and CD36. 3. The composition of any one of the preceding claims wherein, from the ASC population, at least 90% express CD90, CD73, CD13, CD29 and CD166; a maximum of 5% expresses CD45, CD19, CD14 and CD31; a maximum of 10% expresses CD106; between 2 and 15% express CD36; at least 10% express CD146; at least 80% express CD105 and at most 40% express CD34; me at least 90% express CD10, CD140b, CD160, CD204, CD272, CD44, CD54, CD9, Galectin 3, Galectin 9, HLA-G and LTpR; at least 80% express CD49a; at least 60% express CD258 and CD270 and at least 5% express CD200; at most 15% express CD15, CD152, CD163, CD18, CD274, CD39, CD40, CD62L, CD80 and CD86; and a maximum of 30% expresses CXCR4. 4. The composition of any one of the preceding claims wherein, after stimulation with interferon gamma comprising the culture for 3 days in the presence of 50 ng / ml of interferon gamma, the percentage of the ASC population that expresses CD274 increases by less than 80% and the expression level of CD54 in CD54 positive cells increases at least 25-fold compared to a control ASC population cultured for 3 days in the absence of interferon gamma. The composition of any one of the preceding claims, wherein, immediately after thawing, at least 80% of the population of stem cells are viable cells, and the population of stem cells has a doubling of the population of al minus 1 when grown for 48 hours. 6. The composition of any one of the preceding claims, wherein the cryoprotectant comprises Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid), Na +, K +, Ca2 +, Mg2 + 1Cl-, H2PO4 ", HEPES, lactobionate, sucrose, mannitol, glucose, dextran-40, adenosine, and glutathione, and from about 5% to about 15% DMSO. The composition of any one of the preceding claims, wherein the ASC concentration is between 2 x 107 cells and 5 x 107 cells per ml. 8. The composition of any one of the preceding claims, which is a pharmaceutical composition. The composition of any one of the preceding claims, which is frozen. 10. A cell bank comprising a plurality of vials stored under frozen conditions, each vial comprising a composition according to claim 9. The cell bank of claim 10, wherein each vial comprises about 5 ml of the composition, and wherein the cell concentration is in the range of about 2.0 x 107 to about 2.5 x 107 cells per ml. . 12. A process for preparing a pharmaceutical composition comprising a substantially homogeneous population of adult human stem cells, comprising the steps of (i) adding the stromal vascular fraction (SVF) of a lipoaspirate collected from a donor to a bioreactor where at least one surface is pretreated to promote adhesion of adult human stem cells; (ii) in the bioreactor, culturing adherent cells for confluence in serum-free culture medium supplemented with human platelet lysate; (iii) separate adherent cells; (iv) freezing the separated cells in a protein-free cryoprotectant at a concentration of at least 1 x 10 6 cells / ml; (v) thaw the frozen cells and repeat steps (ii) and (iii), and optionally (iv), at least once, (vi) freeze the separated cells at a concentration of at least 1.5 x 107 cells / ml; Y (vii) optionally thawing the frozen composition. 13. The process of claim 12, wherein a) at least one surface of the bioreactor is pretreated with a composition comprising or consisting of cryoprecipitate; b) the culture medium comprises from about 2% to about 15% human platelet lysate; c) the cryoprotectant comprises from about 5% to about 15% of DMSO and Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid), Na +, K +, Ca2 +, Mg2 + 1Cl- , H2PO4 ", HEPES, lactobionate, sucrose, mannitol, glucose, dextran-40, adenosine and glutathione; d) in step (v), thawing of the frozen cells and the repetition of steps (ii) and (iii) is carried out once; or e) a combination of any two or more of (a) to (d). 14. A pharmaceutical composition comprising a suspension of a substantially homogeneous population of adult human stem cells in a protein-free cryoprotectant at a concentration of at least 1.5 x 107 cells per ml, obtained or obtainable according to the process of a any of claims 12 and 13. 15. The composition of any one of claims 1 to 9 and 14 for use as a medicine. 16. The composition of any one of claims 1 to 9 and 14 for use in tissue regeneration, immunosuppression and / or as an anti-inflammatory drug. The composition of any one of claims 1 to 9 and 14 for use in treating or preventing a disease or disorder selected from ischemic tissue disorder, tissue dysfunction or destruction disorder, autoimmune disorder , transplant rejection, and an inflammatory disease or disorder in a subject. The composition for use of claim 17, wherein the disease or disorder is selected from the group consisting of ischemic heart disease, acute myocardial infarction, ischemic stroke, critical limb ischemia, ischemic injury, reperfusion injury. ischemic / primary organ graft dysfunction, intervertebral disc repair, dilated non-ischemic cardiomyopathy, an articular cartilage disorder, Crohn's disease, multiple sclerosis, type 1 diabetes, kidney disease, rheumatoid arthritis, rejection of a transplanted organ, type diabetes 2, arteriopulmonary hypertension and sepsis. The composition for use of claim 18, wherein the disease or disorder is ischemic heart disease, and wherein about 5x107 to about 5x108 cells are administered to the subject in at most about 5 ml by intramyocardial injection. . 20. The composition for use of any one of claims 17 to 19, wherein the subject is not the adipose tissue donor. 21. The composition for use of claim 20, comprising repeating administration to the patient at least once, optionally wherein the adipose tissue is from a different donor.